Rotaviruses are the primary cause of acute dehydrating diarrhea in infants and young children and are responsible for approximately 500,000 deaths worldwide per year. Their impact on human health has stimulated extensive efforts towards the development of rotavirus vaccines by the Laboratory of Infectious Diseases (LID). Large scale phase III trials have shown that a rhesus-based rotavirus tetravalent vaccine developed by the LID is highly effective in protecting against the severe and life-threatening dehydrating diarrhea caused by these viruses. The tetravalent vaccine was approved for use in humans by the FDA, marketed by as Rotashield, and administered to approximately one million children. However, because of concerns that the vaccine may infrequently induce intussusception, the use of the vaccine at the present time has been discontinued. Due to the great impact that rotavirus illness continues to have on human mortality and morbidity, it remains a goal of the Laboratory to develop vaccines against these agents that are effective and safe. The primary goal of this project is the development of a reverse genetics system that can be used to alter the genetic information of the rotavirus genome. Such a system can be used to identify loci within the genome that define the growth characteristics, antigenic properties, and virulence of rotaviruses. With this knowledge, the system can be used to create a new generation of vaccines by the introduction of mutations into the genome of virulent isolates of human rotaviruses. Secondary goals of this project include (i) identification of viral gene products that interfere with innate cellular immune responses, and thereby affect the virulence of the virus, and (ii) analysis of the impact that host cell lines have on the protein coding capacity of vaccine strains of rotavirus. In the past year, progress was made in the production of the large amounts of highly purified recombinant RNAs and functional proteins necessary for attempting the assembly of infectious subviral particles in vitro. Furthermore, information was gained indicating one viral gene product (NSP1) modulates host immune responses by suppressing the function of cellular interferon-response factor 3 (IRF3). An important observation related to this finding was the discovery that the type of cell line used to propagate rotavirus vaccines can have an effect on the ability of the virus to express NSP1.